EP0804571A1 - Spider neurotoxins and method of producing the same - Google Patents

Spider neurotoxins and method of producing the same

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Publication number
EP0804571A1
EP0804571A1 EP95915957A EP95915957A EP0804571A1 EP 0804571 A1 EP0804571 A1 EP 0804571A1 EP 95915957 A EP95915957 A EP 95915957A EP 95915957 A EP95915957 A EP 95915957A EP 0804571 A1 EP0804571 A1 EP 0804571A1
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EP
European Patent Office
Prior art keywords
leu
lys
polypeptide
glu
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95915957A
Other languages
German (de)
English (en)
French (fr)
Inventor
David Robert Bell
Peter Norman Russell Usherwood
Irina Dulubova
Tatiana Volkova
Eugene Grishin
Valery Nelson Institute KRASNOPEROV
Tatiana G. Shemyakin & Ovchinnikov GALKINA
Mikhail V. Shemyakin & Ovchinnikov KHOVOTCHEV
Kirill A. Shemyakin & Ovchinnikov PLUZHNIKOV
Oleg G. Shamotienko
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BTG International Ltd
Original Assignee
BTG International Ltd
British Technology Group Ltd
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Publication of EP0804571A1 publication Critical patent/EP0804571A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates to a novel toxin, and a method of producing a toxin, particularly but not exclusively to an insect specific neurotoxin
  • ⁇ -Latroinsectotoxin ⁇ -LIT
  • ⁇ -Latroinsectotoxins are two such neurotoxins that have been characterised as being vertebrate and invertebrate specific respectively.
  • polypeptide such as a toxin, formed by expression of a truncated form of a gene sequence, or an analogue thereof.
  • the polypeptide is a neurotoxin and preferably corresponds to a toxic derivative of a substantially non-toxic precursor polypeptide encoded by the gene sequence.
  • the polypeptide may comprise an amino acid sequence that corresponds to a truncated form of the amino acid sequence of a substantially non-toxic
  • the amino acid sequence of the polypeptide corresponds to the amino acid sequence of the precursor polypeptide with truncation thereof principally at the carboxy (C) end, and desirably by about 150 to 200 amino acids.
  • the polypeptide amino acid sequence may in addition correspond to the precursor polypeptide amino acid sequence truncated at the amino end (N) preferably by less than 50 amino acids, and desirably by 7 or 28 amino acids.
  • the amino acid sequence of the polypeptide may be homologous to the amino acid sequence of the insect specific neurotoxin ⁇ -Latroinsectotoxin ( ⁇ -LITT or an active derivative thereof, and preferably comprises an amino acid sequence as shown in SEQIDNO1 and SEQIDNO2 or an active derivative thereof.
  • the toxin is expressed from a nucleotide construct or truncated form of the gene sequence comprising a sequence as shown in SEQIDNO1, or active variants thereof.
  • the toxin is expressed from a sequence substantially as provided in a microorganism deposited at The National Collections of Industrial and Marine Bacteria Limited, under Accession No. NCIMB 40632.
  • the invention also provides a protein for use as a toxin comprising an amino acid sequence substantially as shown in SEQIDNO1 and SEQIDNO2, or an active derivative thereof.
  • nucleotide sequence According to a further aspect of the present invention there is provided a nucleotide sequence
  • polypeptide such as a toxin
  • the nucleotide sequence corresponds to a gene encoding for a precursor polypeptide and truncated at the 3' end thereof or an active derivative thereof.
  • the nucleotide sequence corresponds to the gene truncated by about 400 to 650 nucleotide bases, and desirably between 550 to 600 nucleotide bases.
  • the nucleotide sequence may also correspond to the gene truncated at the 5' thereof, preferably by less than 100 nucleotide bases, and desirably by either 84 or 21 nucleotide bases.
  • the nucleotide sequence corresponds to part of a gene encoding for a neurotoxin in the venom of the Black Widow Spider (Latrodectus mactans
  • Tredecimguttatus or an active derivative thereof.
  • the nucleotide sequence may correspond to part of the gene encoding the precursor polypeptide of insect specific toxin ⁇ -Lactoinsectotoxin ( ⁇ -LIT), or an active derivative thereof.
  • the nucleotide sequence preferably codes for a polypeptide comprising a sequence of 991 amino acids.
  • the nucleotide sequence comprises a base sequence as shown in SEQIDNO1, or an active derivative thereof, and preferably as comprised in a microorganism deposited under Accession No. NCIMB 40632 at The
  • nucleotide sequence codes for a polypeptide having an amino acid sequence as shown in SEQIDNO1 and SEQIDNO2, or an active derivative thereof.
  • the nucleotide sequence may be a cDNA derived from mRNA by the use of an enzyme such as reverse
  • the nucleotide sequence may alternatively be an oligonucleotide DNA construct produced perhaps using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • expression system such as a viral or bacterial
  • the polypeptide produced is an active toxin and desirably a neurotoxin substantially as defined above.
  • the truncated form comprises part of a gene which encodes for a non-toxic precursor polypeptide.
  • the truncated form comprises a
  • the expression system comprises E . coli BL21 (DE3) bacterial cells transformed with pT7-7 vectors comprising the truncated form of the sequence, desirably substantially as deposited under Accession No. NCIMB 40632 at The National Collections of Industrial and Marine Bacteria Limited.
  • the expression system may comprise a baculovirus system.
  • a recombinant DNA molecule such as a virus, and in particular a baculovirus comprising a
  • a still further aspect of the present invention provides an expression vector comprising a truncated form of a gene, the truncated form encoding for a toxin generally as defined above.
  • the invention also provides a cell, such as a viral or bacterial cell transformed with a recombinant molecule as defined above.
  • insecticide comprising a toxin as defined above.
  • the insecticide may be so as to be administered orally or topically.
  • the insecticide may comprise a spray.
  • This invention also provides an insecticide system comprising means for expressing a truncated form of a gene to produce a toxin as described above in an insect to kill or incapacitate the insect.
  • the insecticide system may comprise a viral expression system, and desirably a baculovirus expression system.
  • a plant comprising a genetically modified cell containing a truncated form of a gene sequence substantially as defined above.
  • a non-human animal comprising a
  • a toxin formed by processing of a substantially isolated non-toxic precursor
  • the toxin is preferably a neurotoxin and is
  • the toxin amino acid sequence generally corresponds to the amino acid sequence of the precursor polypeptide, truncated by between 150 and 200 amino acids.
  • the toxin amino acid sequence may also be formed by truncation toward the amino (N) end of the precursor polypeptide amino acid sequence, the fragment cleaved therefrom preferably being significantly smaller than the fragment cleaved from the carboxy end, and may comprise 7 or 28 amino acids.
  • the toxin has an amino acid sequence corresponding to polypeptide encoded by part of a gene of the Black Widow Spider (Latrodectus mactans
  • the toxin may comprise or be an analogue of the insect specific neurotoxin
  • the toxin comprises an amino acid sequence as shown in SEQIDNO1 and SEQIDNO2 or an active derivative thereof.
  • a method of producing an active polypeptide from an inactive precursor polypeptide comprising truncating the isolated precursor polypeptide.
  • the isolated precursor polypeptide is truncated at the Carboxyl end, perhaps using proteolytic cleavage, and preferably by site directed mutagenesis. Truncation of the N terminus may also be provided.
  • the active polypeptide is a toxin and is substantially as described above.
  • nucleotide base sequence encoding for a toxin precursor polypeptide as defined above and preferably with an amino acid sequence as shown in SEQIDNO4 or an active derivative thereof.
  • the base sequence preferably comprises the sequence shown in SEQIDNO3 or a derivative thereof.
  • the nucleotide base sequence preferably encodes a precursor polypeptide of the neurotoxin ⁇ -Latroinsectotoxin
  • the base sequence is substantially as provided in the microorganism deposited under
  • a recombinant DNA molecule such as a virus, and more particularly a baculovirus comprising a sequence as defined in the preceding paragraph.
  • the invention provides a cell, such as a bacterial cell or viral cell, transformed with a recombinant DNA molecule as described in the preceding paragraph.
  • This invention also provides an insecticide system comprising means for expressing a gene as described above to produce a precursor polypeptide as described above and to process the precursor polypeptide to produce a toxin in an insect to kill or incapacitate the insect.
  • the insecticide system may comprise a viral expression system, and desirably a baculovirus expression system.
  • a plant comprising a genetically modified cell containing a gene as defined above.
  • non-human animal comprising a genetically modified cell containing a gene as defined above.
  • Preferred embodiments of the present invention will ow be described by way of example only, with reference to the accompanying sequences, in which:-
  • SEQ ID NO. 1 shows the nucleotide base sequence and the corresponding amino acid sequence of a truncated form of a gene and a polypeptide encoded thereby, according to one aspect of the present invention
  • SEQ ID NO. 2 shows the polypeptide sequence of SEQIDNO1
  • SEQ ID NO. 3 shows the nucleotide base sequence and the corresponding amino acid sequence of a gene and a polypeptide encoded thereby, according to another aspect of the present invention.
  • SEQ ID NO. 4 shows the polypeptide sequence of SEQIDNO3.
  • a polypeptide such as a toxin as in SEQIDNO2 is formed by expression of a truncated form of a gene sequence (SEQIDNO1), or an analogue thereof.
  • a toxin from Black Widow Spider (Latrodectus mactans Tredecimguttatus) venom (BWSV), ⁇ -Latroinsecto toxin, ( ⁇ -LIT) has been purified and shown to possess insect specific toxicity.
  • the ⁇ -LIT has been purified and shown to possess insect specific toxicity.
  • the invention includes identification of the sites for cleavage of the precursor protein to produce the toxin, and the precise site of truncation of the gene sequence which has enabled the toxin to be expressed in bacterial, and indeed other suitable hosts.
  • Escherichia coli ( XL - 1 Blue pT7. ⁇ M) cloned with the truncated form of the gene sequence is deposited under Accession No. 40632, and Escherichia coli (HMS 174 pT7. ⁇ FL) cloned with substantially the full gene sequence is deposited under Accession No. 40633).
  • cDNA cloning and sequencing was conducted as follows. Poly(A+)-RNA was isolated from venom glands of the Black Widow Spider (Latrodectus mactans Tredecimguttatus) and a cDNA library constructed in the plasmid vector pSP65 (according to Kiyatkin et al, 1993). A library of 6 ⁇ 10 4 clones was screened with an end-labelled 23-mer oligonucleotide probe based on the
  • N-terminal sequence of ⁇ -LIT (amino acid residues 1-8)- 5' GA(C/T)GA(A/G)GA(A/G)GA(C/T)GG(A/T)GAAATGAC 3' .
  • Hybridization was performed. Positive clones were colony-pun fled and analysed by restriction mapping. The inserts were excised and fragmented by sonication as described (Sambrook et al, 1989) followed by cloning into the Smal site of pBluescript II SK+ and SK- vectors
  • Synthetic primers were used to sequence regions that were not covered by isolated subcloned fragments.
  • DNA and protein sequence analysis was performed using the computer software DNASTAR (Dnastar Inc) and PCGENE ( IntelliGenetics Inc). This work benefitted from the GCG programme mounted on the SERC Daresbury SEQNET facility (Devereux, Haeberli and Smithies, (1984),
  • the full-length cDNA construction was carried out as follows. Two sets of oligonucleotide primers were used to produce N- and C- overlapping parts of ⁇ -LIT coding sequences by polymerase chain reaction. To facilitate subcloning into the expression vector the 5' sense primer (P1) ( TTGGGATCCGATGAAGAAGATGGAGAA ) and 3' antisense primer (P8) ( CAATGGTCGACACAGAAGGAATGGTA ) contained BamHI and SalI restriction enzyme sites.
  • P1 TTGGGATCCGATGAAGAAGATGGAGAA
  • P8 CAATGGTCGACACAGAAGGAATGGTA
  • primers -P9, sense (GTCTGAACCATTTACTGTCC) (position 1283-1302) and P3, antisense ( GTAAGATTACCATCTGCAAC ) (complementary to position 2253-2272) were chosen to produce overlapping fragments with an internal Ncol (2056) restriction site.
  • An oligonucleotide was designed to terminate the protein sequence after amino acid 991-5' CGTTTCGTCGACTCATTCCGGTAAAGTACGACGAAA 3' .
  • the polymerase chain reaction was performed using 1 unit of Taq-polymerase (Promega) under standard conditions (30 cycles, 55°C for 1 min, 72°C for 3 min, 94°C for 1 min, with 100 pmol of each primer and 1-10 ng first-strand cDNA). In the first cycle the de ⁇ aturation time was elongated to 5 min. The molecular mass of the amplified material was checked on an agarose gel. First-strand cDNA synthesis was carried out using First-strand cDNA Synthesis Kit (Pharmacia) with both random and specific primers as recommended by the manufacturer. The PCR products were purified from agarose gel using GeneClean Kit (Bio 101 Inc.), digested with appropriate pairs of enzymes (BamHI and NdeI for the N-terminus part and
  • SEQIONO4 was constructed and designated pT7. ⁇ FL.
  • the first 28 amino acids are believed to be present in the precursor polypeptide in spider venom glands, but cleaved during N-terminal processing.
  • the recombinant protein constitutes approximately 10% of the total bacterial lysate protein.
  • a polyclonal antibody specific was raised to ⁇ -LIT purified from spider venom glands, and demonstrated to be specific for the ⁇ toxin. This protein specifically detected a protein of 130 kDa in bacteria expressing recombinant full-length ⁇ -LIT.
  • ⁇ -LIT had no toxicity towards insects and is considered to be an inactive precursor form of the toxin.
  • ⁇ -LIT purified from venom glands was analysed by mass spectrometry (on a Kratos Kompact MALDI 3 Mass
  • E. coli BL21(DE3) cells transformed with pT7 clones were grown in LB medium containing 100mg ampicillin/ml at
  • Extracts from pT7-7 and pT7.&FL produced no effects on the locusts, but extracts from bacteria carrying pT7. ⁇ M caused rapid lethality.
  • the time of death of the locusts varied from 5 minutes - 4 hours, depending on the potency of the batch of toxin.
  • ⁇ -LIT was applied in standard locust saline (mM: NaCl, 180; KCl, 10; CaCl2, 2; Hepes, 10 (pH 6.8)). A few studies were undertaken using saline in which CaCl2 was omitted. Mechanical responses were recorded using a Grass strain guage connected to a Grass pen recorder. Recordings of miniature excitatory
  • ⁇ -LIT was applied in either standard locust saline, saline in which CaCl2 was omitted or saline which contained MgCl2 substituted for NaCl.
  • the miniature potentials were recorded on video tape and analysed on a MassComp computer using m-house software.
  • Membrane bilayers were formed at the tips of patch pipettes (diam 1-2 ⁇ m; fabricated from Clark
  • Electromedical glass from monolayers of either
  • diphytanoyl phosphat idy lcholine or a mixture of 9 parts isolectin and 1 part cholesterol using a pipette dipping technique (Montal and Muller, 198). Similar patch pipettes were used to excise membrane patches from locust metathoracic extensor tibiae muscle fibres (Huddle et al). In order to reduce the activities of endogenous potassium channels KCl was eliminated from the pipette and bath salines.
  • the current-voltage characteristic of the ⁇ -LITx channel was sigmoidal with a reversal potential at 0mV.
  • the channel was shown to be Ca-selective by manipulating the ionic regimes of patch pipette and bath.
  • a cDNA library from venom gland cDNA was screened with a 23-bp oligonucleotide probe corresponding to the N-terminal sequence of ⁇ -LIT (as described above). To reduce the number of nucleotide ambiguities the codon usage data available from the nucleotide sequences of ⁇ -LT and ⁇ -LIT cDNA (Kiyatkin et al, 1990, Kiyatkin et al 1993) was referred to. Five positive cDNA clones were colony-purified and sequenced. The longest clone (pDT-1) contained more than 2 (kb) of ⁇ -LIT coding region.
  • a PstI-3' fragment was used to rescreen the cDNA library to search for clones encoding the C-terminal part of the toxin.
  • Two overlapping clones, covering the entire open reading frame, have been sequenced in their entirety. The two clones have been demonstrated to be part of a single, continuous RNA from venom glands by polymerase chain reaction across the overlapping region, using two
  • the composite clones encode a cDNA with a frame of 3642 bp starting from the first in-frame Methionine and ending with TAA stop codon
  • ⁇ - LIT was purified to homogeneity from Black Widow Spider venom by three rounds of column chromatography according to (Krasnoperov et al, 1992). 23 amino acid residues of the N-terminal sequence of ⁇ -LIT was sequenced. The pure toxin was digested with trypsin and seven individual peptides were isolated and partially sequenced.
  • Daltons and pi of 5.4 contains all of the peptide sequences determined by amino acid sequencing analysis. There are two in-frame Met residues (-7 and -28) upstream of the N-terminus (as shown in SEQIDNO3) of the mature protein which can serve as translation initiation sites.
  • nucleotide sequence surrounding the ATG codon for Met correlates better with the classical Kozak consensus (Kozak, 1989), but the nucleotide arrangement for Met (-28) strongly corresponds to starting points for at least two other known proteins which have been isolated from arachnids: Major house dust mite allergen (AAAATGA) (Yuuki et al, 1991) and Low molecular weight protein co-purified with ⁇ -Latrotoxin (AAATGA) (Kiyatkin et al, 1992). In both cases, the deduced sequence preceding the N-terminus of the mature protein does not correspond to classical signal peptide structures.
  • sequence of 13 amino acids which precede the first repeat can be viewed as a reduced repeated unit according to its good correlation with a consensus sequence.
  • the majority of ⁇ -LIT repeated units are 33-34 amino acids in length, but two repeats contain 35 (R1) and 36(R6) residues, respectively.
  • the mature protein can be divided into several structural domains: an N-terminus consisting of about 470 amino acid residues and possessing strong linear homology with ⁇ -LIT; the central domain of about 430 amino acids almost completely comprised of tandemly arranged
  • ankyrin-like repeated units and a C-terminal domain of about 160 amino acids.
  • this domain which is absent from ⁇ -LIT, forms a specific region in the primary structure of ⁇ -LIT that has an unusual clustering of Cys-residues and possesses homology with mammalian-specific ⁇ LT (Kiyatkin et al, 1993). So striking structural difference between the two insect-specific neurotoxins suggests that the C-terminal part of the ankyrin-like repeated domain plays a particular role in providing a structural basis for their different functional properties.
  • the high molecular weight protein toxins from the venom of the Black Widow Spider are a potent and specific class of toxins. These toxins offer a great potential for elucidating the function of neural proteins, and for providing insect specific toxins. However this potential has not previously been realised due to the inability to express these protein toxins with any function.
  • the present invention provides for the cloning of a novel DNA transcript encoding for a novel insect-specific toxin, and functional expression of this toxin, and other polypeptides in bacteria.
  • the ⁇ -LIT cDNA was cloned with an oligonucleotide based on the sequence of amino acids 1-23 of the toxin, and its identity confirmed by additional peptide
  • ⁇ -LIT immunochemical identity
  • the deduced primary structure of ⁇ -LIT has considerable similarity to the mammalian specific ⁇ LT and the insect-specific ⁇ -LIT, suggesting that these toxins are part of a family with similar structure.
  • the three proteins have a central domain which is composed of "ankyrin-like" repeats, with 13 repeats in ⁇ -LIT.
  • the ankyrin family of proteins couple spectrin to a variety of integral membrane proteins
  • ⁇ -LIT has no greater similarity to the insect-specific ⁇ -LIT (38%) than to the mammalian- specific ⁇ LT (37%).
  • ⁇ -LIT has only 13 repeats
  • the ⁇ LT and ⁇ -LIT have 19 and 20 ankyrin repeats, respectively.
  • the latter 6/7 repeats have no counterpart in the ⁇ -LIT, and may be a structural unit, as the ⁇ - toxins both contain 6 cysteine residues in this region, with partially conserved spacing.
  • ⁇ -LIT exhibits a marked disparity between the molecular weight of the toxin, as deduced from the cDNA sequence, and the relative mobility of the pure toxin purified from venom. Whilst the N-terminus of the protein has been identified unambiguously by protein sequencing, the precise position of the C-terminus has been difficult to document. Expression of the full length ⁇ -LIT cDNA in bacteria demonstrated that the calculated molecular mass is accurately reflected in the relative mobility of the protein on SDS-PAGE, and that the natural venom derives predominantly, if not wholly from proteolytic, C-terminal processing. The full-length recombinant protein was purified, but was not toxic to locusts under any conditions. The full-length protein is an inactive precursor of the functional toxin.
  • spectrome try which localised the site of cleavage to amino acid 991 of the protein.
  • the cDNA was mutated to produce a protein of 991 amino acids with a sequence as shown in SEQIDNO2, and expressed in bacteria.
  • the mature recombinant protein was soluble and was lethal to
  • a modified plant cell or plant such as a crop plant, containing a recombinant molecule incorporating the truncated sequence.
  • a system comprises a recombinant baculovirus comprising the truncated form.
  • viruses are highly infectious in vivo and resistant to inactivation in host cells, and are capable of high levels of expression of the inserted nucleotide sequence in host insect cells. This is expected to be harmless to the plant and indeed to vertebrates.
  • an insect Upon ingestion of the plant tissue an insect will take in the recombinant molecule and/or toxin, resulting ultimately in the death of the insect. Since the toxin is insect specific, it is expected to have no detrimental effect to humans or animals upon consumption.
  • the invention also provides toxin formed from the expression of a full, isolated gene to produce a
  • precursor polypeptide which is then post-translationally modified.
  • the precursor polypeptide has an amino acid sequence as shown in SEQIDNO4, and the toxin has a sequence as shown in SEQIDNO2.
  • the isolated gene (SEQIDNO3) (or an analogue) encoding for the precursor polypeptide of the toxin ef LIT can be cloned into a vector for expression of the precursor polypeptide.
  • a baculovirus expression system can be used.
  • the precursor polypeptide thus produced can then be truncated at the sites indicated above, by site directed mutagenesis, to produce an active toxin. This enables the toxin ⁇ LIT or an active derivative thereof, to be produced independently of the Black Widow Spider, and thus on an industrial scale, for use as indicated as an insecticide.
  • GGC ATG TAT TCG AAA TTC AGT GAA TGG TCT GAA CCA TTT ACT GTC CAA 1304 Gly Met Tyr Ser Lys Phe Ser Glu Trp Ser Glu Pro Phe Thr Val Gln
  • AAG CCT GTA TCA AAA GTC TTA TGT TCG TAT TTG GAT ACC TTT TCT GAA 3512

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EP95915957A 1994-04-27 1995-04-24 Spider neurotoxins and method of producing the same Withdrawn EP0804571A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9408466 1994-04-27
GB9408466A GB9408466D0 (en) 1994-04-27 1994-04-27 Cloning and functional expression of neurotoxins
PCT/GB1995/000917 WO1995029235A1 (en) 1994-04-27 1995-04-24 Spider neurotoxins and method of producing the same

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EP0804571A1 true EP0804571A1 (en) 1997-11-05

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EP95915957A Withdrawn EP0804571A1 (en) 1994-04-27 1995-04-24 Spider neurotoxins and method of producing the same

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EP (1) EP0804571A1 (ja)
JP (1) JP2000511761A (ja)
KR (1) KR970702916A (ja)
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HU9602959D0 (en) 1997-01-28
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BR9507507A (pt) 1997-09-02
GB2288807A (en) 1995-11-01
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AU2264495A (en) 1995-11-16
CN1147272A (zh) 1997-04-09
WO1995029235A1 (en) 1995-11-02

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